Hello,I´ve performed many RNA extraction of Caco cells. I always get nice ribosomal bands but besides I got a huge band that migrates close below ribosomal 5S (please see attached gel). I don´t know what could it be. I don´t think its a degradation product because it isn´t a smear but a sharp band. I wonder if could that be bacterial RNA. Cells didn´t look contaminated before extraction, but it´s rather possible that a contamination can go unnoticed in caco cells because they expel lot of debris or they could be contaminated with something that cannot be seen under microscope (mycoplasma??)Do you know if bacterial RNA looks like the band that appears in my extraction?

-Marvilla-

Ribosomal RNA from bacteria, although smaller than human still run above 500bp. I think the band at the bottom on your gel is the tRNA...

HTH

-beccaf22-

Agreed -- tRNA.

-HomeBrew-

I don´t agree. If you look closer you´ll see that there are two bands at the bottom of the gel. I have seen in other RNA gels that the intesity of tRNA band is not as much as the big band in my gel.

-Marvilla-

QUOTE (Marvilla @ Oct 14 2005, 12:45 PM)

I don´t agree. If you look closer you´ll see that there are two bands at the bottom of the gel. I have seen in other RNA gels that the intesity of tRNA band is not as much as the big band in my gel.

I thought you said the sharper band is the 5s RNA -- the thick band directly underneath is the tRNA You may be recovering different amounts or get different amount of tRNA expression depending upon cell type, but this band looks just like what I think is tRNA, of course I havent ever confirmed like with a probe or anything, but I would not think it is contamination, I think it is tRNA. I work with Caco cells also and I also see the same band in my RNA gels which my boss and another caco expert from my committee agree it is tRNA. If you want I will send you my picture, let me know...

hope this helps...

-beccaf22-

to me this just looks like a gel effect, nothing more. The whole gel is quite smeary, and with formaldehyde gels you often get "blobs" showing up at the bottom. Also you can see a similar band in the empty well on the right

You can easily check for the presence of tRNA by running the samples on a 15% urea-PAGE gel. The tRNA shows up as a series of discrete bands at around 70nt. It shows up less brightly than the 5S

-John Buckels-

yes it is tRNA. My RNA denaturing gels of mice liver looks exactly the same. I won't worry about it

-asimetrich-

according to my recent experiment, i can tell you it's probably not bacterial RNA.I got a cell culture in which i saw few bacterias on it (the medium was still clear but under microscope it was bacterias). Even if i rinsed the cells by multiple PBS washes, the resulting RNA preparation is a disaster. quite all is degraded. So i assume if you had bacterias in your sample you wouldn't have been able to see the 28 and 18S "so" clear.

-fred_33-

Hi,

Maybe this answer is way too obvious and not as sexy as having bacterial, mycoplasm or alien RNA contaimination, but it is very obvious to me that your RNA is simpply degraded.The ratio of the 28 to 18 S bands should be ~2:1. Yours is not. When this ratio is lost, it is clear that the RNA is degraded. When RNA is degraded there is often a smear/band around the 5S. This can even happen post-purificatin while it is running in the gel (again, from experience). You may disagree, but unfortunately, I have had crappy RNA enough to know that your gel shows claasic "dead" RNA. Some advice, the easiest answer is often the correct one.